JP2666069B2 - Recombinant avian prolactin or recombinant avian preprolactin, recombinant chicken prolactin, recombinant chicken prolactin structural gene, recombinant chicken preprolactin, recombinant chicken preprolactin structural gene, recombinant plasmid, recombinant plasmid Microorganisms containing - Google Patents

Description

The present invention relates to avian prolactin, and particularly to recombinant chicken prolactin, a recombinant chicken prolactin structural gene, a recombinant chicken preprolactin, and a recombinant chicken prolactin. The present invention relates to a chicken preprolactin structural gene, a recombinant plasmid, a recombinant chicken prolactin, and a method for producing a recombinant chicken preprolactin.

[Prior Art] Prolactin is a hormone produced in pituitary eosinophilic cells, is a peptide closely related to growth hormone and placental lactogen, and is considered to be derived from a common ancestral peptide.

Mammalian prolactin is produced in the anterior pituitary gland, but its activity as well as its structure is already known. For example, humans, cows, pigs, sheep, etc. have been reported, and recently, the isolation and structure of fish salmon prolactin has been described by H. Kawauchi et al. In General and Comparative Endocrinology (Ge).
n.Comp. Endcrinol, 49, 446-458, 1983) and Ark Biochemistry and Pyophytics (Arch. B)
iochem. Biophysic, 244 (2), 528-541, 1986), and also by H. Kawauchi et al.
Structure of carp prolactin (Gen. Comp. Endocrinol., 66, 28
0-290, 1987).

It is known that the effects of prolactin in birds are diverse. Maintains so-called maternal behaviors such as crop milk formation, nesting, and incubation, or, in the case of migratory birds, effects on floating at night, irritation, increase in subcutaneous fat (preparing for migration), that is, effects on reproduction or related activities. Wide physiological effects are known.

At present, bird prolactin is considered to have an effect such as inducing maternal behavior in birds, and thus chicken prolactin is expected to be applied particularly in the field of chicken farming.

[Problems to be Solved by the Invention] However, although there is a report that a hormone protein has been isolated from avian prolactin (Scans.
G. et al. Gen. Comp. Endocrinol., 27, 371-379, 1975) There has been no subsequent report, and the structure of hormone proteins and genes has not been analyzed.

Furthermore, at present, natural chicken prolactin is contained only in a trace amount in the chicken pituitary gland and its structural analysis has not been performed. Therefore, its supply is limited because it requires a large amount of pituitary gland.

In view of the above problems, the present inventors have analyzed the structure of chicken prolactin using gene recombination technology, and have reached the present invention for the purpose of mass supply in the future.

Furthermore, according to the present invention, not only is the chicken prolactin structural gene cloned and its nucleotide sequence determined, but also the amino acid sequence of chicken prolactin is elucidated, and chicken prolactin can be produced and isolated by microorganisms such as Escherichia coli. In addition, it is expected that chicken prolactin, which has various physiological actions, will be useful for studying its physiological actions in more detail, and its application will be expanded.

That is, the present inventors isolated the chicken preprolactin gene and determined the structure of the bird prolactin gene, and clarified the structure of chicken prolactin.

[Means for Solving the Problems] The recombinant avian prolactin or the recombinant avian preprolactin according to the first invention of the present invention is an mRNA secreted from an avian pituitary gland containing a polypeptide represented by the following amino acid sequence: Of origin.

The recombinant chicken prolactin according to the second invention is a polypeptide having the following amino acid sequence.

The recombinant chicken prolactin structural gene according to the third invention encodes the amino acid sequence of the second invention.

The recombinant chicken prolactin according to the fourth invention comprises:
It is a polypeptide having the following amino acid sequence.

The recombinant chicken preprolactin structural gene according to the fifth invention encodes the amino acid sequence of the fourth invention.

The recombinant plasmid according to the sixth invention contains a recombinant chicken prolactin structural gene encoding the amide acid sequence of the second invention.

The recombinant plasmid according to the seventh invention contains a recombinant chicken preprolactin structural gene encoding the amino acid sequence of the fourth invention.

A microorganism according to an eighth aspect includes the recombinant plasmid according to the sixth or seventh aspect.

To explain the present invention in more detail, pituitary tissue extracted from chickens is ground in a guanidium thiocyanate solution, and the RNA fraction is collected by centrifugation to obtain the obtained RNA.
The fraction was passed through an oligo d (T) column to concentrate poly (A) ⁺ RNA (messenger RNA) having poly (A) ends.

The enriched poly (A) ⁺ RNA was combined with the ampicillin resistance gene of plasmid pSI4001 and a total length of 25 with 77 d (T) linkages.
Ligated with a 00 base pair cloning primer, a complementary strand DNA of poly (A) ⁺ RNA was synthesized with a reverse transcriptase, and the 3 ′ end of the DNA was transferred with a deoxynucleotide end transferase.
22 d (C) chains were attached.

The end of the cloning vector which does not contain the complementary strand DNA is removed by treatment with the restriction enzyme Hind III. On the other hand, both ends of the prepared cloning vector have Hind III H-adhesive ends and 22 d (G) chains, and the lac promoter 1 is contained therein. A 390 base pair pSI4001-derived linker DNA having a total of 3 frames each consisting of an individual, an SD (Shine-Dalgarno) sequence and a translation initiation codon ATG was bound to this HindIII fragment. At the same time, the DNA chain consisting of the vector primer containing poly (A) ⁺ RNA and the linker is circularized by 22 d (C) and d (G) linkages located at both ends, followed by RNaseH and DNA polymerase. A phosphodiester bond is formed by the treatment, and the chicken prolactin structural gene (complementary chain DN
The DNA strand containing A) was completed as a plasmid having a complete circular DNA structure.

The present inventors introduced the obtained plasmid into Escherichia coli DH1 strain, cultured it at 37 ° C., and grown a colony grown on agar medium containing ampicillin.
250 obtained by treatment of NA restriction enzymes Sty I and Nsi I
And a plurality of positive colonies were obtained by performing colony hybridization using a DNA fragment of 530 base pairs as a probe.

FIG. 1 shows the entire nucleotide sequence of the chicken prolactin structural gene disclosed for the first time by the present invention, and includes the region encoding the amino acid sequence shown in claim 4. From the structure, 30 amino acids considered to be a signal peptide exist at the N-terminal side, and the structure of avian prolactin was elucidated for the first time in the present invention.

Escherichia coli (strain DH1) into which the plasmid pcPRL1 has been introduced as a transformant has been deposited with the Research Institute of Microbial Industry and Technology of the National Institute of Advanced Industrial Science and Technology under the accession number No. 10163 of Microtechnological Laboratory.

To further add, the chicken prolactin structural gene and chicken preprolactin structural gene of the present invention are:
By utilizing this, it is meant that chicken prolactin or chicken prolactin-like polypeptide can be produced in an appropriate host (for example, Escherichia coli, Bacillus subtilis, yeast, etc.). That is, when the gene obtained in the present invention is transformed by introducing a recombinant plasmid containing a gene sequence (eg, a promoter called tac, trp, λPL) which can be regulated in an appropriate host, the polypeptide can be produced. Means that it is under the control of a promoter and can artificially regulate its production.

[Action] The prolactin polypeptide and the derivative thereof according to the present invention are produced by an action such as induction of maternal behavior in birds.
It is expected to be widely used in fields such as breeding and propagation of birds. Since the effects such as induction of maternal behavior in birds are considered,
In particular, chicken prolactin is expected to be applied in the chicken raising field.

EXAMPLES Specific examples of the present invention will be described below.

Example 1. Isolation of total poly (A) ⁺ RNA from chicken pituitary gland: Poly (A) ⁺ RNA was isolated from chicken pituitary gland by the guanidium thiocyanate method (Katsuya Shigesada, Cell Engineering, 2 , 61
6 (1983)).

640 mg (approximately 10 individuals) of the linked pituitary gland of chicken was treated with 6 M guanidium thiocyanate (manufactured by Wako Pure Chemical Industries, Ltd. (hereinafter abbreviated as “Wako”)), 5 mM sodium citrate, 0.55 sarcosyl sodium and 0.1 M β -Crushing with a polytron homogenizer in 5 ml of mercaptoethanol solution,
Solubilized. Pass several times with this homogenate 18G injection needle,
Chromosome-derived DNA was disrupted. This was gently overlaid on a solution of 5.7 M cesium chloride (Wako) and 0.1 MEDTA (pH 8.0) so as to be about twice the amount of this solution, and rotated at 29,000 revolutions with a Beckman SW40Ti rotor. The precipitate of RNA was obtained by centrifugation for 19 hours. This RNA was carefully washed, suspended in 10 mM Tris-HC1, 1 mM EDTA, and ethanol precipitated three times. The obtained precipitate was washed with 10 mM Tris-HC1 (pH 8.
0), suspended in 1 mM EDTA, 0.1% SDS solution, added with LiCl to a final concentration of 0.5 M, incubated at 90 ° C for 2 minutes, and subjected to oligo (dT) cellulose (Collaborative Research) column chromatography. I took it.

The adsorbed poly (A) ⁺ RNA was converted to 10 mM Tris-HC1 (pH 7.5),
Elution was performed with 2 mM EDTA and 0.1% SDS solution. UV light (260n
m) The fraction showing absorption was collected and poly (A) ⁺ RNA6
9 μg was obtained.

Example 2. cDNA synthesis and integration into vector plasmid: cDNA synthesis and integration into a vector were performed by the Okayamaberg method (Molecular and Cellular Biology (Mol. Cell. Biol.), 2.161 (1982); Katsuya Shigesada). Cell Engineering, 2,616 (1983)).

Plasmid pSI4001 (K. Isui et al., Nucleic Acids Res.). 14, 1615 (198
6)) 400 µg of restriction enzyme Kpn I (Nippon Gene,
The following restriction enzymes were all manufactured by Nippon Gene. ) At 37 ° C. for 5 hours to completely digest. After phenol / chloroform treatment, ethanol precipitation is performed and DN
A was recovered. This DNA digested with KpnI is then used to add about 60 poly d (T) chains to the 3 'end of the KpnI-cleaved portion using the reaction of terminal deoxynucleotidyl transferase (TdT, manufactured by Pharmacia). did. After phenol chloroform treatment, ethanol precipitation, recover DNA,
Next, 2.8 Kbp DNA obtained by digestion with restriction enzyme BamHI
Fragments were recovered after separation on a low melting point agarose gel.

The recovered DNA is then subjected to oligo (A) cellulose column chromatography (Collaborative Research).
To obtain a DNA fragment (hereinafter referred to as a vector primer) to which a poly d (T) chain has been sufficiently added.

Preparation of the linker DNA was performed as follows. pS I 400
1, 100 μg was completely digested with restriction enzyme Pst I, genol-chloroform treated, ethanol precipitated, and the Pst
About 1 d of poly d (G) was added to the 3 ′ end of the I cleavage site using TdT.
Added five. Phenol-chloroform treatment, ethanol precipitation, complete digestion of the recovered DNA with the restriction enzyme Hind III, and 5% polyacrylamide gel electrophoresis
A 280-base DNA fragment with poly d (G) was separated and recovered. Hereinafter, this DNA fragment is referred to as linker DNA.

About 5 μg of poly (A) ⁺ RNA prepared in Example 1 and 1.5 μg of vector primer were mixed, 10 units of reverse transcriptase (Wako) was added, and the mixture was reacted at 37 ° C. for 40 minutes. The reaction product was subjected to phenol-chloroform treatment and ethanol precipitation twice, and then reacted with TdT at 37 ° C. for 20 minutes to add about 15 d (C) chains. The reaction product was again subjected to phenol treatment and ethanol precipitation to recover DNA, and the restriction enzyme Hind III
Then, phenol treatment and ethanol precipitation were performed again to recover DNA. Take one tenth of the DNA,
12 ng of the linker DNA prepared as described above was added, and a DNA ligase derived from Escherichia coli (Pharmacia) was added overnight to circularize. E. coli DNA ligase was added to the reaction,
Ribonuclease H (Pharmacia), DNA
Add polymerase I (Nippon Gene) and add 12
Incubate for 1 hour each at 25 ℃ and 25 ℃.
A complete combinatorial plasmid of DNA was made.

Example 3 Selection of Chicken Prolactin cDNA Recombinant Plasmid and Nucleotide Sequence of cDNA Portion
The strain was prepared using the Hanahan method (DNA cloning, vol.
1, p109, IPL PRESS, 1985), and about 10,000 colonies showing ampicillin resistance were fixed on Whatman 541 paper (manufactured by Whatman). Then, a porcine prolactin cDNA was labeled with ³² p by nick translation of a DNA fragment consisting of 250 and 530 bases obtained by treatment with restriction enzymes Sty I and Nsi I, and this was used as a probe in 50% formamide at 42 ° C. When colony hybridization was performed, 8 positive colonies were obtained.

When a recombinant plasmid was extracted from these colonies and analyzed with restriction enzymes, similar results were obtained for all of them. The result is shown in FIG. Then, pcPRL1, which was a clone estimated to be almost full-length cDNA portion, was subjected to the dioxy method (Sanger et al., Proc. Natl. Acad. Sci.) USA, 74. , 5463 (1977)), and the base sequence of chicken prolactin was determined.
FIG. 1 shows the nucleotide sequence and amino acid sequence of the cDNA portion. Tables 1A to 1G show the entire nucleotide sequence of the recombinant plasmid pcPRL1. The base numbers 390 to 1355 in the table are the parts shown in FIG.

[Effects of the Invention] As described above, the present invention is based on the amino acid sequence of chicken prolactin elucidated for the first time by the present invention, and various expression including a deoxyribonucleotide chain encoding the entire or partial amino acid sequence thereof. Vectors are constructed, and each is introduced into Escherichia coli, Bacillus subtilis, photosynthetic bacteria, yeast, animal cells, or the like as appropriate transformants, and then cultured. Thus, recombinant chicken prolactin can be easily obtained from various transformants. There is an effect that a large amount can be obtained.

[Brief description of the drawings]

FIG. 1 is a diagram showing the entire nucleotide sequence of the complementary strand DNA portion containing chicken prolactin and the amino acid sequence revealed from the sequence, and FIG. 2 is a restriction enzyme cleavage map of the portion containing chicken prolactin complementary chain DNA.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification number Agency reference number FI Technical indication C12R 1:19) (54) [Title of the Invention] Recombinant bird prolactin or recombinant bird preprolactin , Recombinant chicken prolactin, recombinant chicken prolactin structural gene, recombinant chicken preprolactin, recombinant chicken preprolactin structural gene, recombinant plasmid, microorganism containing recombinant plasmid

Claims (9)

(57) [Claims] A recombinant avian prolactin or a recombinant avian preprolactin derived from mRNA secreted from the avian pituitary gland, comprising a polypeptide represented by the following amino acid sequence: 2. A recombinant chicken prolactin which is a polypeptide having the following amino acid sequence: 3. A recombinant chicken prolactin structural gene encoding the amino acid sequence of claim 2. 4. A recombinant chicken preprolactin which is a polypeptide having the following amino acid sequence: 5. A recombinant chicken preprolactin structural gene encoding the amino acid sequence of claim 4. 6. A recombinant plasmid containing a recombinant chicken prolactin structural gene encoding the amino acid sequence of claim 2. 7. A recombinant plasmid containing a recombinant chicken preprolactin structural gene encoding the amino acid sequence of claim 4. A microorganism comprising the recombinant plasmid according to claim 6 or 7. 9. The method according to claim 9, wherein the microorganism is Escherichia coli.
A microorganism comprising the recombinant plasmid according to claim 8, wherein the microorganism belongs to